HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral life cycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we performed an unbiased genome-wide screen to identify host factors required for productive HCV infection. Among the identified host factors, we studied SMAD6, an inhibitory SMAD (I-SMAD) of the TGF- signaling pathway. We further investigated the role and mechanism of SMAD6 in modulating HCV infection. We employed various HCV in vitro models to explore the mechanism of action of I-SMADs on HCV infection and characterized cellular signaling pathways regulated by I-SMADs in both hepatoma cell lines and primary human hepatocytes. The expression of I-SMADs and other genes was also studied in HCV-infected hepatocytes and human livers from chronic hepatitis C patients. SMAD6 and its I-SMAD partner SMAD7 enhance HCV entry, predominantly at the viral attachment step, through transcriptionally up-regulation of heparan sulfate proteoglycan (HSPG) expression on cell surface. SMAD6 is also involved in the expression of multiple lipoprotein and cholesterol uptake receptors, including LDLR and SR-BI in hepatocytes. I-SMADs and HSPG levels are elevated in HCV-infected hepatoma cells, primary human hepatocytes and liver biopsies from HCV infected patients. I-SMADs enhance HCV attachment and entry via up-regulation of cellular entry factors including HSPGs, LDLR and SR-BI. HCV infection in turn enhances expression of I-SMADs and downstream signaling pathways as a strategy to facilitate viral propagation. Our study thus reveals a novel I-SMAD-mediated pathway that facilitates HCV entry by transcriptional regulation of hepatic HSPG contents and cholesterol uptake. The transmembrane, lipid-associated protein TM6SF2 is another host dependency factor for HCV in modulating LVP formation and HCV life cycle. HCV co-opts the very low density lipoprotein (VLDL) pathway for morphogenesis, maturation and secretion, and circulates as lipoviroparticles (LVPs). We conducted both TM6SF2 loss-of-function and gain-of-function assays and examined HCV infection in cultured hepatocytes by analyzing viral RNA and protein expression and infectious LVP levels. The density of secreted LVPs was evaluated by iodixanol gradient assay in Huh7.5.1 cells transfected with a plasmid overexpressing tm6sf2 sequence. We measured TM6SF2 expression patterns in liver biopsies from chronic hepatitis C (CHC) patients, livers of HCV infected humanized Alb-uPA/SCID mice, and HCV-infected Huh7.5.1 cells. TM6SF2 depletion in hepatocytes decreased viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatant. In HCV-infected cells, TM6SF2 overexpression enhanced production of infectious LVPs in lower density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice and CHC patient livers. TM6SF2 mRNA levels correlate positively with HCV RNA levels in CHC liver biopsies. SREBP-2 appears to mediate the induction of TM6SF2 expression by HCV. TM6SF2 is requisite for maturation, lipidation and secretion of infectious LVPs; and HCV, in turn, upregulates TM6SF2 expression to facilitate its productive infection. Cellular miRNAs have been shown to regulate hepatitis C virus (HCV) replication, yet a systematic interrogation of the entire repertoire of miRNAs impacting HCV life cycle is lacking. Using the same screening technology, we performed an unbiased strategy to identify cellular microRNAs associated with HCV infection and functionally interrogate these miRNAs with our previous HCV small interference RNA (siRNA) screen database to derive an extensive cellular/viral regulatory network in productive HCV infection. Through genome-wide miRNA mimic and hairpin inhibitor phenotypic screens, and miRNAmRNA transcriptomics analyses, we identified three proviral and nine antiviral miRNAs that interact with HCV in a physiologically relevant manner. These miRNAs were then functionally linked to particular steps of HCV life cycle and relevant viral host dependencies, thereby revealing extensive cellular miRNAmRNA regulatory networks associated with HCV infection and propagation. Further mechanistic studies demonstrated that miR-25, let-7 and miR-130 families repress essential HCV co-factors, thus restricting viral infection at multiple stages. HCV meanwhile subverts the antiviral actions of these miRNAs by downregulating their expression in cell culture and HCV-infected human livers. This comprehensive HCVmiRNA interaction map provides fundamental insights into HCV-mediated pathogenesis and unveils molecular pathways linking RNA biology to viral infections.